Decarboxylation of malonyl substrates is a common enzymatic transformation that occurs in a number of important biological pathways. For example, numerous aerobic and anaerobic bacteria can grow on malonate as the sole carbon source, because they possess malonate decarboxylase systems that transform malonate or malonyl-CoA to acetate.1 Most of these archetypicali malonate decarboxylases are multienzyme systems involving acyl carrier proteins (ACPs) with unusual prosthetic groups as well as biotin dependent decarboxylases.2 Decarboxylation of malonyl units also occurs during the Claisen-type condensation reaction catalysed by β-ketoacyl ACP synthase (KAS) enzyme and domains within fatty acid and polyketide synthases.
An arylmalonate decarboxylase (AMDase) was isolated from the gram-negative bacterium, Alcaligenes bronchisepticus (Bordetella bronchiseptica),3,4 which catalyzes the enantioselective decarboxylation of α-aryl-α-methylmalonates to give optically pure α-arylpropionates. Whilst the biological role of the enzyme has not been established, it has been shown to be a robust catalyst decarboxylating a range of substrates including: phenylmalonic acid 1; 2-methyl-2-phenylmalonic acid 2; 2-methyl-2-naphthyl malonic acid 3 and 2-thienylmalonic acid 4 (see FIG. 1 (a)).5 The AMDase is unusual amongst malonate decarboxylases6 as it does not require biotin or any other co-factors for activity. In addition, decarboxylation does not involve formation of a malonyl thioester-enzyme intermediate7 which is a common driving force for several malonate decarboxylases.1,2 The mechanism of the enzyme is thus of considerable interest. The chiral carboxylic acid products of the AMDase are also potentially attractive for industrial applications, particularly as precursors for pharmaceutical synthesis.
AMDase from B. bronchiseptica (SEQ ID NO:2) shows sequence similarity with a number of racemases and isomerases.7,8 Moreover the putative catalytic cysteine is preserved in all enzymes as shown by sequences alignment. However no AMDase activity was reported for those enzyme and AMDase from B. bronchiseptica (SEQ ID NO:2) remained the only enzyme capable to catalyze the decarboxylation of α-arylmalonates to α-arylpropionates.
In the case of the glutamate and aspartate racemases two cysteine residues are located on either side of the amino acid substrate in the active site. Indeed, it was discussed that the two cysteine residues function in general acid-base catalysis, abstracting the α-proton from the substrate to generate a planar enolate which is re-protonated from the opposite face leading to the racemate.9,10 In the case of the AMDase, a single active site Cys residue (Cys188) has been shown to play a crucial role in enzyme catalysis. This led to the proposal that the mechanism of the AMDase is similar to the racemases. For example, decarboxylation of the substrate 2-methyl-2-phenylmalonate is suggested to result in an enolate anion, which is protonated on the si-face by Cys188 to form a R-configured α-phenylpropionate product.8 To support the mechanistic similarity with the racemase family, the introduction of second cysteine, guided by sequence alignment,8 resulted in a AMDase single point mutant (G74C), which was capable of racemising homochiral α-arylpropionates, albeit with very low catalytic efficiencies.7 In addition the double mutation G74C/C188S, which was predicted to reposition the key active site Cys on the opposite face of the enolate intermediate, led to an enzyme with opposite enantioselectivity. Once again the catalytic efficiency of this mutant was several orders of magnitude lower than the wild type AMDase.11 
WO 2005/07811 provides specific examples of mutants of and AMDase enzyme isolated from B. bronchiseptica KU1201 carrying up to two mutated amino acid residues. The following mutants with altered enzyme activity are suggested: A84G, F85A, A87G, R94A, T103A, 1127A, (F85A, R173A), (F85A, E176A) and (F85A, A178G); The following mutants with improved thermostability are suggested: P15A, P32A, G74A, T75A, D128A, D163A, A165G and C171A. Generically, mutations in at least one of the positions 17, 19, 22, 24, 25, 32, 41, 42, 46, 47, 53, 60, 61, 63, 68, 74, 83, 84, 85, 87, 94, 103, 105, 112, 116, 119, 121, 139, 142, 155, 168, 171, 173, 178, 199, 201, 202, 203, 204, 205, 210, 221, 222, 224, 225, 226, 227, 228, 229, 230, 235, 238, 239 and 240 are mentioned. However, said document neither discloses the entire crystal structure of said mutants or of the parent AMDase or the structural organization of the catalytic site of such enzymes, nor does said document teach or suggest that AMDase activity might be associated with proteins of different origin. The document speculates about further “product structures” which might be accessible via enzyme mutants disclosed therein. However, enzyme activities of a limited number of mutants were merely reported for phenyl malonic acid and alpha-hydroxy-(4-methylphenyl) malonic acid. No hydrocarbyl-substituted, in particular no alkenyl-substituted malonic acid derivatives have been suggested or even tested.
Further enzymes with AMDase activity, so far, have not been described in the art.
There is therefore a need for alternative, optionally improved enzymes with AMDase activity, which potentially are applicable in industrial processes for preparing preferably optically pure decarboxylation products of malonic acid derivatives, as for example α-arylpropionates, and which do not require biotin or any other co-factors for decarboxylase activity.
There is also a need for identifying novel malonate decarboxylase enzyme with extended or altered/modified substrate specificity, i.e. enzymes decarboxylating malonate derivatives different from alpha-aryl malonates, which potentially are applicable in industrial processes for preparing preferably optically pure alpha-substituted monocarboxylic acids from corresponding malonate substrates.
There is also a need for functional mutants derived from such enzymes with AMDase activity or enzymes with extended or altered/modified substrate specificity, which potentially are applicable in industrial processes for preparing preferably optically pure mono-carboxylic acids from malonate substrates. Said functional mutants may be adapted to the preferred or exclusive decarboxylation of malonate substrates different from aryl malonates, as for example non-aromatic alpha-substituted malonate derivatives, like alkenyl-substituted malonates.
It is therefore an object of the present invention to provide such alternative or optionally improved enzymes with malonate decarboxylase activity (MDase activity) as mentioned above.